Understanding the factors that structure freshwater invertebrate assemblages is essential for predicting biodiversity patterns across heterogeneous landscapes. Here, we provide the first comprehensive assessment of ostracod diversity across multiple freshwater ecoregions of Botswana, a semi-arid country encompassing a wide range of hydrological and climatic conditions. We analyzed ostracod assemblages from 19 sites, integrating spatial, regional, and local environmental variables with biotic predictors derived from macro-, meio-, and microinvertebrate communities. Multivariate analyses revealed that spatial variables, strongly correlated with bioclimatic gradients, were the dominant predictors of ostracod assemblage composition. These gradients reflected broad-scale climatic differences among ecoregions and explained substantially more variation than local physical and chemical water properties. Biotic interactions, represented by taxonomic and functional composition of associated invertebrates and vegetation type, also significantly contributed to model performance, highlighting their previously underappreciated role in shaping ostracod assemblages. Local abiotic environmental factors had comparatively weak explanatory power. Our findings demonstrate that ostracod diversity in Botswana is primarily structured by large-scale spatial-bioclimatic patterns, with additional influence from biotic interactions. This study provides new insights into the ecological drivers of freshwater microcrustaceans in semi-arid regions and establishes a baseline for future biodiversity assessments in southern Africa.